Nucleic acid molecules encoding bank1 splice variants

ABSTRACT

The present invention relates to a new splice variant of BANK1, the use of SNPs in BANK1 for diagnostics and the use of antagonists to modulate BANK1 and/or the BANK1 pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/738,418, filed Apr. 16, 2010, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2008/065980,filed Nov. 21, 2008, which claims the benefit of U.S. Provisional PatentApplication No. 61/004,480, filed Nov. 28, 2007, the disclosures ofwhich are hereby incorporated by reference in their entirety, includingall figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Mar. 25, 2010 and is 29 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a new splice variant of BANK1, the useof SNPs associate with BANK1 for diagnostics and the use of antagoniststo modulate BANK1 and/or the BANK1 pathway.

BACKGROUND OF THE INVENTION

Genetic techniques allow the identification of single nucleotidepolymorphisms (SNPs) in individuals. SNPs are changes in a gene in onesingle nucleotide. Identification of SNPs can be correlated with abiological pathway having implications for a particular disease. Thepolymorphisms may be correlated also with a predisposition or risk for adisease by application of statistical analyses. Accordingly, targeting aparticular biological pathway related to a disease is a means to treatsuch disease.

B-cell scaffold protein with ankyrin repeats (BANK1) is expressed in Bcells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR)stimulation. The BANK1 gene has 284 kb. BANK1 is an adaptor protein (6,7) expressed mainly in B cells. The two full length isoforms of 785 and755 amino acids, differ by 30 amino acids in the N-terminal region codedby the alternative exon 1A (FIG. 1 e) and contain ankyrin repeat motifsand coiled-coil regions—structures highly similar between BANK1, BCAPand Dof adaptor proteins (8). B cell activation through BCR engagementleads to tyrosine phosphorylation of BANK1, which in turn promotes itsassociation with the protein tyrosine kinase Lyn and the calcium channelIP3R (3). BANK1 serves as a docking station bridging together andfacilitating phosphorylation and activation of IP3R by Lyn and theconsequent release of Ca²⁺ from endoplasmic reticulum stores (3, 9). Itwas previously found that IP3R associates with the SNP rs10516487 lyingwithin a region essential for binding of IP3R.

The BANK1 SNPs rs17266594 and rs3733197 have also been described in theliterature.

None of the above SNPs have been described in the literature to beuseful for the prediction of an inflammatory, auto-immune orneurological disease.

BANK1 and the pathway it is involved in, is considered to haveimplications for inflammatory and auto-immune disorders. Inparticularly, BANK1 is expressed in B-cells and therefore the pathwaywherein BANK1 is involved has an implication for diseases associatedwith B-cells, e.g. Systemic Lupus Erythematosus (SLE). MultipleSclerosis (MS) is related to T-cells, however, also the role of B-cellshas been discussed in this disease. Accordingly, polymorphisms in theBANK1 gene may be used to diagnose a predisposition or risk for MS.Moreover, the BANK1 pathway may have implications for MS. Inconsequence, targeting this pathway and its modulation may represent ameans to prevent or treat MS.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a novel splice variant ofBANK1 is provided.

According to another aspect of the invention, a method is provided fordiagnosing an individual for the predisposition of, the risk ofdeveloping or suffering from an auto-immune or inflammatory diseasewherein the pathway of BANK1 is involved.

According to another aspect of the invention, a method for the treatmentand/or prevention of an auto-immune or inflammatory disease is providedusing an antagonist targeting BANK1, the biological pathway of BANK1and/or factors connected to the BANK1 pathway.

According to another aspect of the invention, a method of treatingdiseases is provided wherein the pathway of BANK1 is involved using anantagonist targeting BANK1, the biological pathway of BANK1 and/orfactors connected to the BANK1 pathway.

BRIEF DESCRIPTION OF THE SEQUENCES AND DRAWINGS

SEQ ID NO: 1, 3, 5 are the nucleic acid sequences of the BANK1 delta 2splice variant of human, chimpanzee and mouse, respectively.

SEQ ID NO: 2, 4, 6 are the amino acid sequences of the BANK1 delta 2splice variant of human, chimpanzee and mouse, respectively.

FIGS. 1 a-1 e. Association of rs17266594 with increased levels of thefull-length isoform of BANK1. (a) Total expression of BANK1 gene inseparated mononuclear cell subpopulations. (b) RT-PCR of the coding partof BANK1 amplified from total human spleen cDNA reveals two bands on agel. 1 kb ladder (New England Biolabs) is shown on the left. Theidentity of both bands, 2.3 kb upper band and 1.9 kb smaller band, wasconfirmed by sequencing analysis. (c) Relative mRNA expression levels ofthe full-length and delta 2 isoforms of BANK1, as determined byquantitative real-time RT-PCR on total RNA purified from human PBMCs.Data represent mean±S.D. 39 individuals with TT for the branch pointsite SNP, 34 with TC and 10 with CC genotype were analysed. Full-lengthtranscript: TT versus CC, P=0.0004 (Student's t-test); delta 2transcript: TT versus CC, P=0.0088. (d) Total BANK1 expression was notsignificantly affected by SNP rs17266594. (e) Schematic structure of the5′-end of the gene. SNP rs17266594 located in the branch point site ofintron 1 alters splicing efficiency of the full-length and delta 2transcripts. SNP rs10516487 results in non-synonymous substitution ofArg₆₁ to His. Alternative splicing gives rise to two isoforms,full-length and delta 2 with in-frame deletion of entire exon 2 ofBANK1. Thus, the short protein isoform lacks the putative domain forIP3R binding and could function as a dominant negative isoformattenuating signaling from the full-length protein.

IP3R BD-inositol 1,4,5-triphosphate receptor binding domain, LynBD-tyrosine kinase Lyn binding domain.

FIG. 2 a. Linkage disequilibrium and haplotype block structure acrossBANK1. Data calculated with Haploview analysis of our data using theSwedish cases and controls run for 30 SNPs across the gene.

FIG. 2 b. R2 for all SNPs across BANK1.

FIG. 2 c FIGS. 2 a and 2 b (combined)

FIG. 3. Frequencies of the haplotypes constructed with rs17266594 andrs10516487 (74.1% TG, 24.2% CA), and allele frequencies for rs3733197(68.0% G, 32.0% A). The figure also shows the frequencies of thehaplotypes when including all three SNPs (64.1% TGG, 10.1% TGA, 20.3%CAA, 3.8% CAG). Data is calculated using all populations, combined.

DETAILED DESCRIPTION OF THE INVENTION

The following paragraphs contain definitions used according to theinvention and are intended to apply uniformly throughout thespecification and claims unless otherwise expressly set out definitionprovides a broader definition.

The present invention is directed to an isolated nucleic acid sequencecomprising the sequence of BANK1 lacking exon 2. In a preferredembodiment the nucleic acid is of human, chimpanzee, or mouse origin. Asa reference for the BANK1 sequence one may refer to Nature 431 (7011),931-945 (2004).

In the human BANK1 sequence as described in NCBI's human genome assemblybuild 36, chromosome 4 the exons/introns are as follows:

Exon1: 102930919-102931130 Intron 1: 102931131-102969987 Exon2:102969988-102970386 Intron2: 102970387-102995214 Exon3:102995215-102995369 Intron3: 102995370-103002705 Exon4:103002706-103002844 Intron4: 103002845-103010684 Exon5:103010685-103010824 Intron5: 103010825-103035484 Exon6:103035485-103035590 Intron6: 103035591-103058172 Exon7:103058173-103058369 Intron7: 103058370-103161693 Exon8:103161694-103161772 Intron8: 103161773-103165380 Exon9:103165381-103165689 Intron9: 103165690-103170139 Exon10:103170140-103170445 Intron10: 103170446-103184018 Exon11:103184019-103184087 Intron11: 103184088-103200390 Exon12:103200391-103200569 Intron12: 103200570-103203254 Exon13:103203255-103203318 Intron13: 103203319-103211454 Exon14:103211455-103211484 Intron14: 103211485-103212524 Exon15:103212525-103212580 Intron15: 103212581-103213863 Exon16:103213864-103213928 Intron16: 103213929-103214184 Exon17:103214185-103214918

It is preferably possible that only part of the BANK1 exon 2 is deleted.Such a molecule is equally useful according to the invention.

In one embodiment the isolated nucleic acid comprises SEQ ID NO: 1, 3,or 5, or the complement of said nucleic acid sequence.

In one embodiment the invention relates to an isolated nucleic acidwhich:

-   -   a) hybridizes under high stringency conditions; or    -   b) exhibits at least about 85%, preferably at least about 90%        and more preferably at least 95% identity over a stretch of at        least about 30 nucleotides        with a nucleic acid selected from the group consisting of SEQ ID        NO: 1, 3, or 5, or a complement of said nucleic acid sequence.

Another embodiment of the invention is a polypeptide encoded by any ofthe nucleic acid sequences as mentioned above.

Another embodiment is a vector comprising a nucleic acid as describedabove, preferably a nucleic acid selected from the group consisting ofSEQ ID NO: 1, 3, or 5, or a complement of said nucleic acid sequence.

Preferably the vector containing said nucleic acid molecule isoperatively linked to at least one expression control sequence allowingexpression in prokaryotic or eukaryotic host cells of the encodedpolypeptide.

Another embodiment is a host cell transformed with a vector or a nucleicacid as described above.

Yet another embodiment of the invention is a method for making apolypeptide as described above comprising culturing a host cell asdefined above under conditions in which the nucleic acid is expressed,and recovering the polypeptide encoded by said nucleic acid from theculture.

Another embodiment is a method for genotyping comprising the steps of:

-   -   a. Isolating a nucleic acid from a sample of an individual; and    -   b. Determining whether in rs10516487 a guanine or an adenine is        present, in rs17266594 a thymine or a cytosine is present, in        rs3733197 an adenine or a guanine is present in the biallelic        marker.

In a preferred method the identity of the nucleotides at said biallelicmarkers is determined for both copies of said biallelic markers presentin said individual's genome.

The method for genotyping according to the invention is preferablyperformed by a microsequencing assay. The method preferably furthercomprises amplifying a portion of a sequence comprising the biallelicmarker prior to said determining step. Preferably said amplifying isperformed by PCR. The method according to the invention furthercomprises the step of correlating the result of the genotyping stepswith a risk of suffering or a predisposition for an auto-immune diseaseor inflammatory disease.

In a preferred embodiment the method is performed, wherein the presenceof a guanine in rs10516487, a thymine in rs17266594 and an adenine inrs3733197 in said individual indicates that said individual suffersfrom, has a predisposition for or is at risk of suffering from saidauto-immune disease or inflammatory disease.

The method of the invention preferably is applied wherein the disease isSystemic Lupus Erythrematosus or Multiple Sclerosis.

Now that the inventors have established the association between BANK1and SLE and MS or related diseases, it should be understood thatadditional susceptibility alterations can be identified within said geneor polypeptide, e.g., following the methodology disclosed in theexamples.

The presence of an alteration in the BANK1 gene may be detected by anytechnique known per se to the skilled artisan, including sequencing,pyrosequencing, selective hybridisation, selective amplification and/ormass spectrometry including matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) (Gut et al., 2004). In aparticular embodiment, the alteration is detected by selective nucleicacid amplification using one or several specific primers. The alterationis detected by selective hybridization using one or several specificprobes.

Further techniques include gel electrophoresis-based genotyping methodssuch as PCR coupled with restriction fragment length polymorphismanalysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing; fluorescent dye-based genotyping technologies such asoligonucleotide ligation assay, pyrosequencing, single-base extensionwith fluorescence detection, homogeneous solution hybridization such asTaqMan, and molecular beacon genotyping; rolling circle amplificationand Invader assays as well as DNA chip-based microarray and massspectrometry genotyping technologies (Shi et al., 2001).

Furthermore, RNA expression of altered genes can be quantified bymethods known in the art such as subtractive hybridisation, quantitativePCR, TaqMan, differential display reverse transcription PCR, serial,partial sequencing of cDNAs (sequencing of expressed sequenced tags(ESTs) and serial analysis of gene expression (SAGE)), or parallelhybridization of labeled cDNAs to specific probes immobilized on a grid(macro- and microarrays and DNA chips. Particular methods includeallele-specific oligonucleotide (ASO), allele-specific amplification,fluorescent in situ hybridization (FISH) Southern and Northern blot, andclamped denaturing gel electrophoresis.

Protein expression analysis methods are known in the art and include2-dimensional gel-electrophoresis, mass spectrometry and antibodymicroarrays (Freeman et al., 2004 and Zhu et al., 2003).

Sequencing can be carried out using techniques well known in the art,using automatic sequencers. The sequencing may be performed on thecomplete gene or, more preferably, on specific domains thereof,typically those known or suspected to carry deleterious mutations orother alterations.

Amplification may be performed according to various techniques known inthe art, such as by polymerase chain reaction (PCR), ligase chainreaction (LCR) and strand displacement amplification (SDA). Thesetechniques can be performed using commercially available reagents andprotocols. A preferred technique is allele-specific PCR.

Nucleic acid primers useful for amplifying sequences from the BANK1 geneare able to specifically hybridize with a portion of the BANK1 gene thateither flanks or overlaps with a susceptibility alteration. The primersequence overlaps with the alteration when said alteration is containedwithin the sequence of the BANK1 gene to which the primer hybridizes.The primer sequence flanks the alteration when the primer hybridizeswith a portion of the BANK1 gene that is preferably located at adistance below 300 by of said alteration, even more preferably below250, 200, 150, 100, 50, 40, 30 or 20 by from said alteration.Preferably, the primer hybridizes with a portion of the BANK1 gene thatis at 5, 4, 3, 2, 1 by distance or immediately adjacent to saidalteration.

In another embodiment the method for detecting whether an individual hasa predisposition for or is at risk of an auto-immune disease orinflammatory disease according to the invention comprises the steps:

-   -   a. Isolating the nucleic acid of an individual;    -   b. Detecting and quantifying the BANK1 full length nucleic acid;    -   c. Detecting and quantifying the BANK1 delta 2 nucleic acid;    -   d. Determining the ratio b./c. and/or c./b. of the results of        step b. and c.

In this method the nucleic acid is preferably a mRNA, cRNA or cDNA.

In step d. of the above method the determined ratio is an indication ofthe disease or its increased susceptibility. The more full length mRNAand the less delta 2 splice variant the more risk of disease anindividual has. In particular, the higher this ratio is in the b./ccorrelation and the lower this ratio is in the c./b. correlation thehigher is the risk to develop an auto-immune or inflammatory diseases,in particular SLE or MS.

The inventors have found that the total BANK1 mRNA is not influenced bythe presence of particular SNPs. IN particular SNPs rs10516487,rs17266594 and rs3733197 do not change the total amount of BANK1 mRNAcontent. Accordingly the ratio of full length to delta 2 splice variantof BANK1 mRNA or cDNA is not influenced by the presence of the SNPs ofthe invention. Preferably the ratio b./c. or c./b is about 1. The ratiosuseful in the invention are as described above either b./c. or c./b.

A change in rs17266594 from TT to TC to CC has an influence in theamount of delta 2 BANK1 splice variant mRNA detectable. A ration ofb./c. greater than 1, preferably significantly greater than 1 isindicative of a suffering from, or a predisposition for auto-immune orinflammatory diseases, preferably Systemic Lupus Erythrematosus orMultiple Sclerosis. A ration of c./b. less than 1, preferablysignificantly less than 1 is indicative of a suffering from, or apredisposition for auto-immune or inflammatory diseases, preferablySystemic Lupus Erythrematosus or Multiple Sclerosis. A change in thisSNP from TT to CC may be most reliably be used to make this prediction.The suffering or predisposition may be expressed by calculation of theodd ration (OD). It will be appreciated by the skilled person that anymethod detecting and/or calculating a change in the SNP rs17266594and/or mRNA or cDNA of BANK1 full length and/or delta 2 splice variantmay be used to detect a predisposition for auto-immune or inflammatorydiseases. In one embodiment the invention may be applied by comparingthe mRNA of the BANK1 delta 2 splice variant of a sample with a control.The control may be chosen from one sample or a number of pooled samples.

The SNPs rs10516487 and rs3733197 can also be used to predict asuffering or predisposition and may serve as indirect markers. Accordingto the invention also other SNPs may be used as predictive markers if alinkage with the above markers can be detected. Such a linkage,preferably strong linkage, is expressed by the LD and is preferably D′0.7, preferably D′ 0.8, more preferably D′ 0.9. Such markers can beidentified by standard techniques known in the art.

In another embodiment the invention relates to a method for thetreatment and/or prevention of diseases selected from auto-immune orinflammatory diseases using an antagonist targeting BANK1, thebiological pathway of BANK1 and/or factors connected to the BANK1pathway. Preferably disease is Systemic Lupus Erythrematosus or MultipleSclerosis.

The antagonist may be any molecule that antagonizes partly oressentially completely the targets of interest. Preferably theantagonist targets BANK1, LYN and/or IP3R or their interaction.Preferably the antagonist targets the nucleic acid of BANK1. In oneembodiment the antagonist is an anti-sense RNA, siRNA, an Aptamer, apeptide or a small molecule. In another embodiment the antagonist is anantibody or antibody fragment specifically binding to the targets BANK1,LYN and/or IP3R. Particularly preferred is an antagonist that bindsspecifically to IP3R or interferes with the function of IP3R. In thismanner it can be preferably achieved that the impact of B-cells involvedin the disease development or manifestation of the disease is positivelymodulated, preferably inhibited.

The preferred SNPs as used in the invention are as follows:

Biallelic marker Alternative nucleotides rs10516487 G/A rs17266594 T/Crs3733197 A/G

The risk allel of rs10516487 is G. The risk allel of rs17266594 is the Tand of rs3733197 is A. It will be understood that also other SNPs inLinkeage Disequilibrium (LD) may be used in the sense of the inventionas described herein.

All references cited in this application are herewith incorporated byreference. In the following the present invention shall be illustratedby means of the following examples, which are not construed to be viewedas limiting the scope of the invention.

EXAMPLES

A set of 279 Swedish cases with SLE and 515 Swedish controls weregenotyped for the 100 k Affymetrix SNPs array. After filtering, datafrom 85042 SNPs was used. As our purpose was to identify non-MHC genesand important functional polymorphisms, we proceeded to perform ananalysis of the genomic location of the associated SNPs within knowngenes, discarding genomic deserts. Analysis of the data showed thatamong all the non-MHC-associated SNPs, one (rs10516487) was anon-synonymous substitution of arginine to histidine (triplet cGc->cAc,Arg->His) at amino-acid position 61 (from exon1A) of the BANK1translated protein (allelic association, P=6.4×10⁻³; genotypicassociation, P=2.01×10⁻²). This SNP was ranked as #679 across the wholegenome scan in the allelic association analysis and as #2148 in thegenotypic test. The estimated FDR (False Discovery Rate) was 71.1% and77.5% for these selections, respectively (2). Four more SNPs withinBANK1 showed also association with SLE in the Affymetrix scan(Supplementary Table 1). The described B cell-specific expression ofBANK1 and its potential role in B cell receptor-mediated activation ledus to pursue this gene (3, 4).

We genotyped 30 SNPs in Swedish cases and 352 controls including theAffymetrix SNPs covering the complete 284 kb of the BANK1 gene. Two SNPswere not polymorphic in our population. Individual SNP analysis showedthat 9 SNPs including rs10516487 were associated (Table 1). Using thesolid-spine LD (Linkage Disequilibrium) haplotype block definitionavailable from Haploview, 5 LD blocks could be recognized. All of theSNPs showing genetic association were lying on block 2, 3 and 4. Nogenetic association was detected for SNPs located in block 5 (Table 1,Supplementary Table 2 and FIG. 2 a).

To confirm the genetic association, we genotyped four more sets of casesand controls from Germany, Spain, Italy and Argentina for rs10516487. Wecould corroborate the genetic association with all the European sets,although the Argentine set showed a clear tendency without reachingsignificance (Table 2). We performed homogeneity and combinabilityanalysis of the sets using the Breslow-Day method. As the data could becombined, a meta-analysis was performed on all the sets comprising 3971individuals. The Mantel-Haenzel (MH) test revealed a P value reachinggenome-wide significance and a pooled odds ratio of 1.38 (X²=39.243,P=3.74×10¹⁰, 95% CI 1.25-1.53) for the allelic association. Asignificant genotypic association was also observed (Table 2).

We initiated a detailed analysis of BANK1 expression and structure. Weobserved that indeed and as described, BANK1 is primarily expressed inCD19+ B cells at high levels, while very low expression could bedetected in CD4+, CD8+ and CD14+ cells (FIG. 1 a). We then sequenced theproximal promoter region, exon1A, exon1B, and exon2 (where haploblock 2is located) and 500 by up and downstream of these exons in 24 SLEpatients and 8 controls. No novel SNPs were found for these regions. Inorder to clone BANK1 cDNA in an expression vector for functionalanalysis, we amplified full-length cDNA with distal primers.Surprisingly, two bands were detected on a gel after PCR (FIG. 1 b).Subsequent cloning and sequencing revealed a new isoform with anin-frame deletion of the entire exon 2 (delta 2 isoform of BANK1). Weanalyzed cDNA from 83 healthy individuals and 30 SLE patients and foundthat this isoform was present in each sample, indicating that it isconstitutively spliced. Moreover, this isoform was detected by PCRamplification of cDNA from chimp and mouse spleen as well, suggestingits conserved expression across species. Thus, we detected transcriptsfor three BANK1 isoforms, two full-length using exon1A or exon1B and adelta 2 isoform.

We next performed quantitative analysis of isoform expression inperipheral blood mononuclear cells. First, the relative levels of thetwo full-length isoforms, beginning with exon 1A and exon 1B, weredetermined. Since the latter transcript was present at very low levels,we continued the analysis measuring common full-length isoform levels.We noticed that the ratio of the full-length (FL) isoform to delta2 wasnot constant, which would be expected if delta 2 were equally expressedregardless of the genotypes of the analyzed samples. On the contrary,samples could be divided into groups according to the FL/delta 2 isoformratio. After close examination of the genomic sequences surrounding exon2 where putative signals affecting splicing could be located, one SNP,rs17266594, was found to lie in the putative branch point site and couldpotentially affect splicing. When expression data was re-groupedaccording to this SNP, a clear difference between the genotypes could beobserved (FIG. 1 c). Individuals homozygous for the T allele and thushaving the classical structure of the branch point site (5) (YNYTGAYYN),showed equal expression of both isoforms, while expression of thefull-length transcript was significantly suppressed (up to 40%) withconcomitant upregulation of delta 2 isoform expression in individualshomozygous for the minor allele C. Total BANK1 transcription level wasnot significantly affected by the SNP (FIG. 1 d). Genotyping of all ofour sets of cases and controls for rs17266594 showed that the T allelewas associated with SLE (Table 2; P=4.74×10⁻¹¹, OR=1.42; 95% CI1.28-1.58).

Both SNPs, rs17266594 and rs10516487, are separated by 153 nucleotides(nt) and are in strong LD (D′=0.95; R2=0.90; FIG. 2 b). The T allele ofthe first SNP and the G allele of second one were found in the same riskhaplotype associated with SLE (Table 2, bottom; P=4.75×10⁻⁶; OR 1.30,95% CI 1.16-1.45) and FIG. 3.

We identified five non-synonymous substitutions in the databases. Whilemost SNPs were non-polymorphic, one, rs3733197, an alanine to threoninesubstitution in amino acid position 383 (triplet Gca->Aca) in exon 7coding for the ankyrin repeat-like motif, showed association in thecombined sample (X²=16.576; P=4.67×10⁻⁵ (OR=1.23, 95% CI 1.11-1.36;)although it had not shown association in our first analysis on Swedishindividuals nor in the whole Scandinavian set (Table 1 and SupplementaryTable 3). This SNP is in haploblock 4 (FIG. 2 a) 88211 bp apart fromrs10516487 (D′=0.72; R2=0.39) and rs17266594 (R2=0.27), could segregatewith the risk haplotype composed of the other two SNPs in some cases(FIG. 3) and could be a minor functional polymorphism.

Thus, herein we identify three functional polymorphisms in BANK1associated with SLE.

The associated T allele of rs17266594 correlates with increased levelsof the full-length isoform of BANK1. Thus, both polymorphisms incombination would lead to the achievement of one effect—high expressionof a “more active” protein—through more efficient splicing of thefull-length transcript that encodes a protein with an arginine residuein the IP3R binding domain. Since the delta 2 isoform lacks the entireexon 2 coding for IP3R binding and PH domains, it possibly functions asa dominant negative isoform thereby attenuating BANK1-mediated signaling(FIG. 1 e).

Importance of mutations in ankyrin motifs for interaction with IP3R wasrecently highlighted by the discovery linking single amino acidsubstitutions in the adaptor protein ankyrin-B with cardiac arrhythmiaand sudden cardiac death (10). While the alanine is associated with SLE,the rare allele A of rs3733197 might create a potential site forthreonine kinases (11).

B cells are the major cell type affected in SLE. Novel therapies areaimed at depleting hyperactivated B cells that may function not solelyas autoantibody producing cells, but also as important regulators of theinnate and adaptive immune responses through antigen presentation andcytokine-mediated signaling (12). Functional and expressionabnormalities of signaling molecules in B cells have been described inlupus. Of particular interest is the fact that Lyn, a binding partner ofBANK1 is of key importance in human and mouse lupus autoimmune disease(13-18).

B cell hyperresponsiveness or a lack of control of B cell activationduring immune responses. The precise role of BANK1 in BCR-mediatedsignaling remains unclear since two reports published so far containconflicting data regarding the stimulatory or inhibitory role of BANK1on B cell activation. Given the previously unreported existence of thealternative splicing of exon 2 we can speculate that the negative rolefor BANK1 assigned for the KO model was in part because of the remainingexpression of the delta 2 isoform, as this exon was targeted by theKO-construct (4).

DNA samples

279 cases and 515 controls were genotyped for the 100 k array. Of theseindividuals 279 cases and 352 controls were typed for the BANK1 coverageshown in Table 1.

For the functional polymorphisms an additional 185 Swedish patients weregenotyped and 465 of the controls were available for genotyping ofrs17266594 and rs3733197. We also added for the final MH (MantelHaentsel) analysis and OR (Odds Ratio) estimation 84 Danish cases withthe Swedish cases comprising the Scandinavian set shown in Table 2. Thereplication sets included 384 North German patients and 374 controls,288 Argentine patients and 372 controls, 286 Italian patients and 252controls. The Spanish cohort included 799 patients and 542 controls fromseveral regions in Spain. 707 of the patients and 469 of the controlswere genotyped for rs10516487 and rs3733197, and 678 of the patients and457 of the controls for rs17266594. The reason for this is that DNA froma number of controls was not available. The German, Spanish andArgentine patients have all been previously described (19). The Italiancases are a multicenter collection of patients and their matchedcontrols from Rome, Siena, Milan and Naples, that is North andMid-Italy. All patients fulfil the 1982 ACR (American College ofRheumatology) criteria for the classification of SLE (20).

Genotyping

Genotyping of the 100 k Affymetrix array was performed according to themanufacturers instructions. Fine mapping and replication for SNPsrs10516487, rs17266594 and rs3733197 were done using TaqMan SNPgenotyping assays (Applied Biosystems, Foster City, Calif.). TheAffymetrix genotyping and fine mapping were performed at Serono GeneticsInstitute in Evry, France (now MerckSerono SA). The functionalpolymorphism replications were done. One hundred and six of samples weregenotyped twice for verification showing 100% concordance. Genotypingsuccess rate for all the samples was over 92%.

Statistical Analysis

For the 100K Affymetrix whole-genome scan analysis, pre-processingfilters have been applied: SNPs have been discarded if (i) theproportion of missing genotypes is higher than 5%, (ii) the relativeminor allele frequency is lower than 1% or (iii) the probability thatthe observed genotype distribution results from sampling a SNP whichfollows the Hardy-Weinberg equilibrium is lower than 0.02. Only SNPsfrom autosomal chromosomes have been kept for the sake of homogeneitybetween male and female individuals. SNP sequences have been mapped ontoNCBI 36 human genome assembly and SNPs with multiple localizations havebeen discarded. For each remaining SNP, genotypic and allelicfrequencies in cases and controls are calculated and the correspondingprobability values are computed using exact (non-asymptotic) andunbiased algorithms (21). The False-Discovery Rate (FDR) is thenestimated using the method described by Former, et al. (2).

For fine mapping analyses, genetic association, haplotype estimation, LDand R2 were all estimated using Haploview (v4.0RC2). The Breslow-Daytest of combinability and the Mantel-Haenzel test were performed usingthe StatsDirect software (v2.4.6). As the Breslow-Day test showedcombinability of the strata, the MH test for fixed effects was used inthe analysis. Haplotypes were estimated using the PHASE software (v2.1)(22, 23). Genotypic odds ratios were calculated using the Unphasedsoftware (v3.0.9) (24).

Sequencing

DNA fragments for sequencing were amplified with the correspondingprimers (see Supplementary Table 4), purified from agarose gel withQIAquick gel extraction kit (Qiagen) and sequenced using BigDyeTerminator 3.1 (Applied Biosystems) at the Uppsala Genome Center.

RNA Purification and BANK1 Expression Analysis

Total RNA was purified with TRIZOL Reagent (Invitrogen) from peripheralblood mononuclear cells (PBMCs) obtained with agreed consent fromhealthy donors and lupus patients. 2 μg of RNA were reverse-transcribedwith 2 U of MultiScribe transcriptase in PCR buffer II containing 5 mMMgCl₂, 1 mM dNTPs, 0.4 U of RNase inhibitor and 5 μM oligo-dT. Allreagents were purchased from Applied Biosystems. cDNA synthesis wasperformed at 42° C. for 80 min, and then the reaction was terminated at95° C. for 5 μM. All cDNA samples were diluted to 15 ng/μl.

BANK1 expression was determined by real-time PCR on an ABI PRISM 7700Sequence Detector (Applied Biosystems) with SDS 1.9.1 software. TotalBank1, both alternative full-length isoforms and delta2 isoform werequantified with SYBR Green and relevant primers (see Supplementary Table4). We performed initial denaturation at 95° C. for 5 mM followed by 45cycles of PCR (95° C. for 15 s, 62° C. for 15 s and 72° C. for 30 s).PCR buffer provided with enzyme was supplemented with 3 mM MgCl₂, 200 μMof each of dNTPs, primers, SYBR Green (Molecular Probes), 15 ng of cDNAand 0.5 U of Platinum Taq polymerase (Invitrogen). Expression levelswere normalized to the levels of TBP in the same samples amplified withcommercial reagents (Applied Biosystems). All experiments were run intriplicate. Independent cDNA synthesis was carried out twice.

Cloning of Human, Mouse and Chimpanzee BANK1 Delta 2 Isoform

Purification of total RNA from mouse spleen and cDNA synthesis wereconducted as described above for the human PBMCs. Total RNA fromchimpanzee (Pan troglodytes) spleen was kindly provided by Drs. TomasBergström and Lucia Cavelier, Uppsala University. Human gene wasamplified from Human Spleen BD Marathon-Ready cDNA (Clontech). Afterinitial denaturation at 95° C. for 5 min, 35 cycles (95° C. for 20 s,60° C. for 15 and 72° C. for 2 min 30 s) were performed in PCR buffercontaining 2 mM MgSO₄, 200 μM of each of dNTPs, 0.4 μM of each of thecorresponding primers (see Supplementary Table 4), and 0.5 U of PlatinumTaq-High Fidelity enzyme (Invitrogen). Chimp cDNA was amplified withhuman-specific primers. PCR products were purified from agarose gel andcloned in pCR 4-TOPO vector (Invitrogen) according to the manufacturer'sinstructions. Plasmid DNA from positive clones was purified with QIAprepSpin Miniprep kit (Qiagen) and verified by sequencing.

Accession Codes

BANK1 delta 2 transcripts were deposited in Genbank under the followingaccession numbers EU051376 for human, EU051377 for chimpanzee andEU051378 for mouse.

URLs. Haploview: www.broad.mit.edu/mpg/haploview/; GraphPad Software:http://www.graphpad.com; Protein analysis: http://www.ebi.ac.uk/saps/;http://smart.embl-heidelberg.de/, http://ca.expasy.org/prosite/,http://www.cbs.dtu.dk/services/NetPhos/.

TABLE 1 Association of SNPs in BANK1 in Swedish SLE SNP rs Associatedname allele Chi Sq P Value rs7675129 T 0.147 0.701  rs11726012 G 0.4950.4963 rs11097755 C 0.406 0.524  rs4522865 A 4.758 0.0292 rs4496585 A1.933 0.1644 rs4572885 T 4.442 0.0355 rs10516487 G 7.185 0.0074rs10516486 C 10.041 0.0015 rs17200824 A 2.780 0.0955 rs6849308 C 7.3470.0067 rs10516482 C 8.709 0.0032 rs10516483 C 9.121 0.0025 rs10516484 A0.577 0.4476 rs4493533 C 0.833 0.3614 rs3733197 A 0.006 0.9402 rs2631271G 6.793 0.0092 rs2850390 C 1.032 0.3096 rs2631265 T 0.001 0.9815rs2631267 G 0.048 0.827  rs2631268 T 1.375 0.2409 rs10516491 C 2.3880.1223 rs1872701 G 1.454 0.2278 rs2850393 T 0.313 0.5759 rs2850396 C0.344 0.5575 rs10516490 G 0.311 0.5769 rs10516489 T 0.312 0.5712rs10516488 G 0.537 0.4635 rs1395306 T 1.739 0.1872

SUPPLEMENTARY TABLE 1 BANK1 SNPs in the 100k Array SNP rs numberPosition (−log) P value SNP_A-1701374 rs10516487 103108254 2.27SNP_A-1701494 rs10516486 103108454 2.79 SNP_A-1664926 rs6849308103133261 2.22 SNP_A-1706628 rs10516482 103137348 2.52 SNP_A-1744756rs10516483 103149083 3.25 SNP_A-1683131 rs2631271 103271574 n.s.SNP_A-1697391 rs10516489 103331537 n.s.

TABLE 2 Genotypic, Allelic and Haplotypic Association of rs10516487(R61H) and rs17266594 in five sets of SLE cases and controls and jointanalysis with Mantel-Haenz Population GG GA AA Chi square P-Value Oddsratio (CI) a Allele G Allele A P-Value Odds ratio (CI) rs10516487Scandinavian SLE Cases (536) 309 (57.6%) 200 (37.3%) 27 (5.0%) 11.78740.0028 GG: 2.12 (1.29-3.47) 818 (76.3%) 254 (23.7%) 7.27E−04 1.39(1.14-1.68) Controls (565) 276 (48.8%) 238 (42.1%) 51 (9.0%) GA: 1.59(0.96-2.63) 790 (69.9%) 340 (30.1%) Argentina SLE Cases (255) 164(64.3%)  75 (29.4%) 16 (6.3%) 3.8013 0.1495 GG: 1.41 (0.73-2.72) 403(79%)   107 (21%)   0.0564 1.31 (0.98-1.74) Controls (337) 190 (56.4%)121 (35.9%) 26 (7.7%) GA: 1.01 (0.51-2.00) 499 (74.3%) 173 (25.7%)Germany SLE Cases (312) 181 (58.0%) 118 (37.8%) 13 (4.2%) 11.8503 0.0027GG: 2.60 (1.32-5.14) 480 (76.9%) 144 (23.1%) 8.13E−04 1.52 (1.18-1.95)Controls (360) 166 (46.1%) 163 (45.3%) 31 (8.6%) GA: 1.73 (0.87-3.44)495 (68.8%) 225 (31.2%) Italy SLE Cases (279) 166 (59.5%) 100 (35.8%) 13(4.7%) 7.5139 0.0234 GG: 2.49 (1.22-5.09) 432 (77.4%) 126 (22.6%) 0.00781.46 (1.09-1.94) Controls (245) 123 (50.2%)  98 (40.0%) 24 (9.8)   GA:1.88 (0.91-3.91) 344 (70.2%) 146 (29.8%) Spain SLE Cases (702) 414(59.0%) 243 (34.6%) 45 (6.4%) 11.3579 0.0034 GG: 1.26 (0.77-2.06) 1071(76.3%)  333 (23.7%) 0.0065 1.30 (1.07-1.58) Controls (446) 219 (49.1%)197 (44.2%) 30 (6.7%) GA: 0.82 (0.50-1.35) 635 (71.2%) 257 (28.8%)Pooled Cases (2003) 1187 (59.3%)  706 (35.2%) 110 (5.5%)  3080 (76.9%) 926 (23.1%) 3.74E−10 1.38 (1.25-1.53) c Controls (1968) 974 (49.9%) 817(41.8%) 162 (8.3%)  2763 (70.8%)  1141 (29.2%)  Population TT CT CC Chisquare P-Value Odds ratio (CI) Allele T Allele C P-Value Odds ratio (CI)rs17266594 Scandinavian SLE Cases (511) 296 (57.9%) 189 (37.0%) 26(5.1)   9.4399 0.0089 TT: 2.17 (1.28-3.66) 781 (76.4%) 241 (23.6%)0.0036 1.36 (1.10-1.68) Controls (416) 210 (50.5%) 166 (39.9%) 40 (9.6%)CT: 1.75 (1.03-2.99) 586 (70.4%) 246 (29.6%) Argentina SLE Cases (274)188 (68.6%)  77 (28.1%)  9 (3.3%) 14.1697 8.38E−04 TT: 3.26 (1.51-7.06)453 (82.7%)  95 (17.3%) 1.06E−04 1.73 (1.30-2.31) Controls (346) 192(55.5%) 124 (35.8%) 30 (8.7%) CT: 2.07 (0.93-4.59) 508 (73.4%) 184(26.6%) Germany SLE Cases (241) 132 (54.8%)  98 (40.7%) 11 (4.6%) 7.71640.0211 TT: 2.46 (1.19-5.09) 362 (75.1%) 120 (24.9%) 0.0080 1.43(1.09-1.87) Controls (335) 151 (45.1%) 153 (45.7%) 31 (9.3%) CT: 1.81(0.87-3.76) 455 (67.9%) 215 (32.1%) Italy SLE Cases (231) 130 (56.3%) 87 (37.7%) 14 (6.1%) 10.1706 0.0062 TT: 2.42 (1.19-4.93) 347 (75.1%)115 (24.9%) 0.0016 1.59 (1.18-2.14) Controls (219)  92 (42.0%) 103(47.0%)  24 (11.0%) CT: 1.45 (0.71-2.97) 287 (65.5%) 151 (34.5%) SpainSLE Cases (678) 404 (59.6%) 231 (34.1%) 43 (6.3%) 14.8617 5.93E−04 TT:1.04 (0.62-1.76) 1039 (76.6%)  317 (23.4%) 0.010 1.29 (1.06-1.56)Controls (458) 225 (49.1%) 208 (45.4%) 25 (5.5%) CT: 0.65 (0.38-1.09)658 (71.8%) 258 (28.2%) Pooled Cases (1856) 1102 (59.4%)  655 (35.3%) 99(5.3%) 2859 (77.0%)  853 (23.0%) 4.74E−11 1.42 (1.28-1.58) c Controls(1774) 870 (49.0%) 754 (42.5%) 150 (8.5%)  2494 (70.3%)  1054 (29.7%) Population TG/TG TG/other other/other Chi square P-Value TG otherP-Value Odds ratio (CI) Haplotype Scandinavian SLE Cases (509) 293(57.6%) 190 (37.3%) 26 (5.1%) 4.6600 0.0973 776 (76.3%) 242 (23.8%)0.22738 1.14 (0.91-1.43) Controls (365) 205 (56.2%) 128 (35.1%) 32(8.8%) 538 (73.8%) 192 (26.4%) Argentina SLE Cases (260) 187 (71.9%)  65(25.0%)  8 (3.1%) 11.8483 0.0027 439 (84.4%)  81 (15.6%) 0.00032 1.72(1.27-2.36) Controls (317) 189 (59.6%) 103 (32.5%) 25 (7.9%) 481 (75.9%)153 (24.1%) Germany SLE Cases (237) 131 (55.3%)  94 (39.7%) 12 (5.1%)6.6099 0.0367 356 (75.1%) 118 (24.9%) 0.01228 1.40 (1.07-1.85) Controls(331) 151 (45.6%) 150 (45.3%) 30 (9.1%) 452 (68.3%) 210 (31.7%) ItalySLE Cases (230) 130 (56.5%)  87 (37.8%) 13 (5.7%) 9.4922 0.0087 347(75.4%) 113 (24.6%) 0.00225 1.57 (1.16-2.13) Controls (214)  92 (43.0%) 99 (46.3%)  23 (10.7%) 283 (66.1%) 145 (33.9%) Spain SLE Cases (589)324 (55.0%) 217 (36.8%) 48 (8.1%) 5.4954 0.0641 865 (73.4%) 313 (26.6% 0.43109 1.09 (0.88-1.34) Controls (374) 186 (49.7%) 165 (44.1%) 23(6.1%) 537 (71.8%) 211 (28.2%) Pooled Cases (1825) 1065 (58.4%)  653(35.8%) 107 (5.9%)  2783 (76.2%)  867 (23.8%) 4.75E−06 1.30 (1.16-1.45)Controls (1601) 823 (51.4%) 645 (40.3%) 133 (8.3%)  2291 (71.5%)  911(28.5%) a Genotypic odds ratio calculated using homozygosity for theprotective allele as reference with OR = 1 ^(b)Mantel-Haenzel Chi squareusing fixed effects c Using the Robins, Breslow and Greenland method

SUPPLEMENTARY TABLE 2 SNP rs number MB Build 36 Location in BANK1rs7675129 102894046 intergenic rs11726012 102925041 promoter rs11097755102928331 5′UTR rs4522865 102934911 intronic rs4496585 102937309intronic rs4572885 102954536 intronic rs10516487 102970099 exon coding(NS)* rs10516486 102970299 exon 2 (synonymous) rs17200824 102971612intronic rs6849308 102995106 intronic rs10516482 102999193 intronicrs10516483 103010928 intronic rs10516484 103011108 intronic rs4493533103039707 intronic rs3733197 103058310 exon coding NS rs2631271103133419 intronic rs2850390 103163019 intronic rs2631265 103164099intronic rs2631267 103167495 intronic rs2631268 103167753 intronicrs10516491 103171889 intronic rs1872701 103172704 intronic rs2850393103174239 intronic rs2850396 103187471 intronic rs10516490 103193084intronic rs10516489 103193382 intronic rs10516488 103196800 intronicrs1395306 103204873 intronic *NS: non-synonymous substitution

SUPPLEMENTARY TABLE 3 Genotypic and Allelic Association of rs3733197 infive sets of SLE cases and controls and joint analysis withMantel-Haenzel test. Population GG GA AA Chi square P-Value Odds ratio(CI) a Scandinavian SLE Cases (419) 167 (39.9%) 192 (45.8%) 60 (14.3%)1.2365 0.5389 GG: 1.04 (0.69-1.58) Controls (444) 163 (36.7%) 220(49.6%) 61 (13.7%) GA: 0.89 (0.59-1.33) Argentina SLE Cases (287) 177(61.7%)  97 (33.8%) 13 (4.5%)  9.6496 0.0080 GG: 2.36 (1.20-4.66)Controls (363) 184 (50.7%) 147 (40.5%) 32 (8.8%)  GA: 1.62 (0.81-3.25)Germany SLE Cases (272) 128 (47.1%) 112 (41.2%) 32 (11.8%) 4.1431 0.1260GG: 1.65 (1.01-2.69) Controls (362) 148 (40.9%) 153 (42.3%) 61 (16.9%)GA: 1.40 (0.85-2.28) Italy SLE Cases (253) 131 (51.8%) 102 (40.3%) 20(7.9%)  8.2595 0.0161 GG: 1.74 (0.92-3.29) Controls (251)  98 (39.0%)127 (50.6%) 26 (10.4%) GA: 1.04 (0.55-1.98) Spain SLE Cases (588) 307(52.2%) 234 (39.8%) 47 (8.0%)  3.4580 0.1775 GG: 1.14 (0.72-1.82)Controls (455) 212 (46.6%) 206 (45.3%) 37 (8.1%)  GA: 0.89 (0.56-1.43)Pooled Cases (1819) 910 (50.0%) 737 (40.5%) 172 (9.5%)  Controls (1875)805 (42.9%) 853 (45.5%) 217 (11.6%)  Population Allele G Allele A Chisquare P-Value Odds ratio (CI) Scandinavian SLE Cases (419) 526 (62.8%)312 (37.2%) 0.301 0.5832 1.06 (0.87-1.29) Controls (444) 546 (61.5%) 342(38.5%) Argentina SLE Cases (287) 451 (78.6%) 123 (21.4%) 9.787 0.00181.15 (0.95-1.40) Controls (363) 515 (70.9%) 211 (29.1%) Germany SLECases (272) 368 (67.6%) 176 (32.4%) 4.297 0.0382 1.28 (1.00-1.63)Controls (362) 449 (62.0%) 275 (38.0%) Italy SLE Cases (253) 364 (71.9%)142 (28.1%) 6.696 0.0097 1.42 (1.08-1.87) Controls (251) 323 (64.3%) 179(35.7%) Spain SLE Cases (588) 977 (72.1%) 379 (27.9%) 2.099 0.1474 1.50(1.15-1.96) Controls (455) 630 (69.2%) 280 (30.8%) Pooled Cases (1819)2686 (70.4%)  1132 (29.6%)  16.5763 4.67E−05 1.23 (1.11-1.36) Controls(1875) 2463 (65.7%)  1287 (34.3%) 

SUPPLEMENTARY TABLE 4 PRIMER SEQUENCES SEQ SEQ Gene/gene ID IDfragment/isoform Forward NO Reverse NO hBANK cDNA CACCTCAACCGCCAC  7ATAATAACCTTCT  8 amplification AATGCTGCCAGCA TTAATGATCTTTC TTGCTotal BANK1 AGAGGAAACTACACC  9 GATGAGTTCTTCC 10 qRT-PCR TTACATAGCTCTGACCATCAG Total full-length TCAAAGCAGATGGGA 11 isoforms GATCTCAACDelta2 isoform CAGCGCCCCCAGATT 12 CTGAAG Exon1A full-lengthCAGCGCCCCCAGGAA 13 isoform ATACA Alternative GCCTATTCTTTGTTTT 14exon1 full-length GGAAATACA isoform Common reverse primer for allisoforms for qRT- PCR CACATGGAATTTC 15 AGTGGGAAGCAC Common reverseprimer for gel-analysis ATCACAGTAGACA 16 TTGACATGGAC

For Genomic Sequencing:

Gene/gene SEQ ID SEQ ID fragment/isoform Forward NO Reverse NOpromoter, exon TTGGAGAGGGTAT 17 AAGCAGGGCTAC 18 1A and 5′-part ofTTAGAGCCATA CAATTCACCAG intron1 Alternative CTATGATACTGGA 19AGCATATGACCA 20 exon1B AATACTGTCAGT GCTGATCAG Exon2 TTGATTTACTATG 21TTACATAAGAAA 22 AAAATATCAAGC CCAGCTTCCAG mouse BANK1 ACCTCCCGCAATG 23ACATGGAATTTCC 24 Cdna CTTCCTGT CCAGGAAGCAC

REFERENCE LIST

-   1. Sherer, Y., Gorstein, A., Fritzler, M. J. & Shoenfeld, Y. (2004)    Semin Arthritis Rheum 34, 501-37.-   2. Former K, L. M., Guedj M, Dauvillier J and Wojcik J. Hum Hered,    In Press.-   3. Yokoyama, K., Su Ih, I. H., Tezuka, T., Yasuda, T., Mikoshiba,    K., Tarakhovsky, A. & Yamamoto, T. (2002) Embo J 21, 83-92.-   4. Aiba, Y., Yamazaki, T., Okada, T., Gotoh, K., Sanjo, H.,    Ogata, M. & Kurosaki, T. (2006) Immunity 24, 259-68.-   5. Burge, C. B., Tuschl, T. & Sharp, P. A (1999), ed. Gesteland, R.    F., Cech, T. R. & Atkins, J. F (Cold Spring Harbor Laboratory Press,    Cold Spring Harbor, N.Y.), pp. 525-560.-   6. Jordan, M. S., Singer, A. L. & Koretzky, G. A. (2003) Nat Immunol    4, 110-6.-   7. Kurosaki, T. (2002) Nat Rev Immunol 2, 354-63.-   8. Okada, T., Maeda, A., Iwamatsu, A., Gotoh, K. &    Kurosaki, T. (2000) Immunity 13, 817-27.-   9. Patterson, R. L., Boehning, D. & Snyder, S. H. (2004) Annu Rev    Biochem 73, 437-65.-   10. Mohler, P. J., Schott, J. J., Gramolini, A. 0., Dilly, K. W.,    Guatimosim, S., duBell, W. H., Song, L. S., Haurogne, K., Kyndt, F.,    Ali, M. E., Rogers, T. B., Lederer, W. J., Escande, D., Le Marec, H.    & Bennett, V. (2003) Nature 421, 634-9.-   11. Blom, N., Gammeltoft, S. & Brunak, S. (1999) J Mol Biol 294,    1351-62.-   12. Anolik, J., Sanz, I. & Looney, R. J. (2003) Curr Rheumatol Rep    5, 350-6.-   13. Liossis, S. N., Kovacs, B., Dennis, G., Kammer, G. M. &    Tsokos, G. C. (1996) J Clin Invest 98, 2549-57,-   14. Huck, S., Le Corre, R., Youinou, P. & Zouali, M. (2001)    Autoimmunity 33, 213-24.-   15. Liossis, S. N., Solomou, E. E., Dimopoulos, M. A., Panayiotidis,    P., Mavrikakis, M. M. & Sfikakis, P. P. (2001) J Investig Med 49,    157-65.-   16. Hibbs, M. L., Harder, K. W., Armes, J., Kountouri, N., Quilici,    C., Casagranda, F., Dunn, A. R. & Tarlinton, D. M. (2002) J Exp Med    196, 1593-604.-   17. Flores-Borja, F., Kabouridis, P. S., Jury, E. C.,    Isenberg, D. A. & Mageed, R. A. (2005) Arthritis Rheum 52, 3955-65.-   18. Cornall, R. J., Cyster, J. G., Hibbs, M. L., Dunn, A. R.,    Otipoby, K. L., Clark, E. A. & Goodnow, C. C. (1998) Immunity 8,    497-508.-   19. Kozyrev, S. V., Lewén, S., Ling a Reddy, M.V.P., Pons-Estel, B.    A., The Argentine Collaborative Group, Witte, T., The German    Collaborative Group, Junker, P., Laustrup, H., Gutiérrez, C.,    Suárez, A., González-Escribano, M. F., Martín, J., The Spanish    Collaborative Group and Alarcón-Riquelme, M. E. (2007) Arthritis and    Rheumatism 56, 1234-41.-   20. Tan, E. M., Cohen, A. S., Fries, J. F., Masi, A. T., McShane, D.    J., Rothfield, N. F., Schaller, J. G., Talal, N. &    Winchester, R. J. (1982) Arthritis Rheum 25, 1271-7.-   21. Guedj, M., Wojcik, J., Della-Chiesa, E., Nuel, G. &    Former, K. (2006) Hum Hered 61, 210-21.-   22. Stephens, M. & Donnelly, P. (2003) Am J Hum Genet 73, 1162-9.-   23. Stephens, M., Smith, N. J. & Donnelly, P. (2001) Am J Hum Genet    68, 978-89.-   24. Dudbridge, F. (2003) Genet Epidemiol 25, 115-21.-   25. Freeman, W. M. and S. E. Hemby (2004). “Proteomics for protein    expression profiling in neuroscience.” Neurochem Res 29(6): 1065-81.-   26. Gut, I. G. (2004). “DNA analysis by MALDI-TOF mass    spectrometry.” Hum Mutat 23(5): 437-41.-   27 Shi, M. M. (2001). “Enabling large-scale pharmacogenetic studies    by high-throughput mutation detection and genotyping technologies.”    Clin Chem 47(2): 164-72.-   28. Zhu, H. and M. Snyder (2003). “Protein chip technology.” Curr    Opin Chem Biol 7(1): 55-63.

1. A method for genotyping comprising the steps of: a) isolating anucleic acid from a sample of an individual; and b) determining inB-cell scaffold protein with ankyrin repeats (BANK1) encoding nucleicacid whether: biallelic marker rs10516487 is present as a guanine or anadenine, biallelic marker rs17266594 is present as a thymine or acytosine, and/or biallelic marker rs3733197 is present as an adenine ora guanine.
 2. The method according to claim 1, wherein the nucleotidesat said biallelic markers are determined for both copies of saidbiallelic markers present in said individual's genome.
 3. The methodaccording to claim 1, wherein said determining is performed by amicrosequencing assay.
 4. The method according to claim 1, furthercomprising amplifying a portion of a sequence comprising the biallelicmarker prior to said determining step.
 5. The method according to claim4, wherein said amplifying is performed by PCR.
 6. The method accordingto claim 1, further comprising the step of correlating the result of thegenotyping steps with a risk of suffering or a predisposition for anauto-immune disease or inflammatory disease.
 7. The method according toclaim 1, wherein the presence of a guanine in rs10516487, a thymine inrs17266594 and a thymine in rs3733197 in said individual indicates thatsaid individual suffers from, has a predisposition for or is at risk ofsuffering from said auto-immune disease or inflammatory disease.
 8. Themethod according to claim 7, wherein the disease is Systemic LupusErythrematosus or Multiple Sclerosis.
 9. A method for detecting whetheran individual has a predisposition for or is at risk of an auto-immunedisease or inflammatory disease comprising the steps: a) isolating thenucleic acid of an individual; b) detecting and quantifying the BANK1full length nucleic acid; c) detecting and quantifying the BANK1 delta 2nucleic acid; and d) determining the ratio b./c. and/or c./b. of theresults of step b) and c).
 10. The method according to claim 9, whereinthe nucleic acid is a mRNA, cRNA or cDNA.
 11. A method for the treatmentof diseases selected from auto-immune or inflammatory diseasescomprising the administration of an antagonist targeting BANK1, thebiological pathway of BANK1 and/or factors connected to the BANK1pathway.
 12. The method according to claim 11, wherein the disease isSystemic Lupus Erythrematosus or Multiple Sclerosis.
 13. The methodaccording to claim 11, wherein the antagonist targets BANK1, LYN and/orIP3R or their interaction.
 14. The method according to claim 13, whereinthe antagonist targets the nucleic acid of BANK1.
 15. The methodaccording to claim 11, wherein the antagonist is an anti-sense RNA,siRNA, an aptamer, a peptide, an antibody or fragment thereof or a smallmolecule.